The occurrence of tick-borne pathogens in dogs on communal land in Botswana was studied using Reverse Line Blot (RLB). Eighty blood samples were collected from dogs that underwent a spay and neuter program in Maun, Botswana. These were sent to the Department of Veterinary Tropical Diseases, University of Pretoria, and were stored at the Molecular Laboratory. DNA was extracted from the samples followed by the Polymerase Chain Reaction (PCR) and the RLB. Six samples were found to be positive for Ehrlichia canis. Two of the samples were positive for a concurrent infection of E. canis and Ehrlichia ruminantium. There was one sample that was positive for the non-pathogenic Ehrlichia sp. Omatjenne. Although six samples were positive for the Babesia/Theileria catch-all probe, none hybridised with any of the species-specific probes. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Veterinary Tropical Diseases / unrestricted

This thesis focuses on the molecular characterization of tick-borne parasites of dogs in South Africa. Emphasis is placed on Babesia, Erhlichia, incidental and novel parasite infections that may cause morbidity or mortality in infected dogs. An outbreak of canine babesiosis in the Netherlands is also reported in this thesis. Molecular techniques were employed to isolate, amplify and characterize genomic DNA of these parasites to species level. During preliminary screening of blood samples collected from various sites in the country, that included the Onderstepoort Veterinary Academic Hospital, SPCAs and private clinics throughout seven provinces in South Africa, it was discovered that domestic dogs harboured a wide variety of tick-borne pathogens. The most frequently encountered parasites in South Africa were Babesia rossi, a novel Theileria species of dogs, B. vogeli and Ehrlichia canis respectively. The parasites occurred as single or mixed infections. Incidental infections that included B. gibsoni and Trypanosoma congolense were also detected using PCR. Although it was anticipated that zoonotic Ehrlichial infections of dogs would be detected, none were found. Babesia vogeli was reported for the first time in South Africa although, without any clinical significance. An outbreak of autochthonous canine babesiosis in the Netherlands was confirmed to have been caused by Babesia canis. Dermacentor reticulates was implicated in the transmission of the parasite to naïve dogs. Clinical significance of B. rossi and the novel Theileria sp. of dogs was evaluated. Babesia rossi was found to be of significant clinical importance. Genotyping of B. rossi isolates revealed that parasite genotypes could be correlated to disease phenotype. Additionally, specific genotypes could also be associated with fatalities. Although the characterization of the Theileria sp. in dogs was a first report in South Africa, the clinical significance of this infection in dogs appeared to be poorly resolved. The dangers of having non-endemic species becoming established in South Africa was highlighted with the incidental finding of a B. gibsoni infection in an imported dog. The results of this thesis have shown therefore that populations of dogs that live in tickendemic areas are exposed to single or multiple tick-borne pathogens. These pathogens continue to cause morbidity and mortality in susceptible dogs. Correct diagnosis (supported by molecular diagnostic tools) followed by appropriate treatment offers a better understanding and management of these tick-borne pathogens. Preventative measures should be fully evaluated and applied to prevent these tick-borne pathogens from adversely affecting the canine population in South Africa and elsewhere. / Thesis (PhD)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted

The incidence of tick-borne zoonoses such as Ehrlichiosis, Rocky Mountain Spotted Fever, and Lyme disease has steadily increased in the southeastern United States in recent years. According to the Centers for Disease Control and Prevention (CDC), the southeastern states accounted for 1,200 of the 27,000 total cases of Lyme disease reported in the U.S. in 2007. Although Ixodes scapularis is the most commonly recognized vector for the Lyme disease spirochete Borrelia burgdorferi, Dermacentor variabilis (a common vector for Rocky Mountain Spotted Fever) also has been shown to be a viable host for this pathogen. The purpose of the present study was to use PCR and DNA sequencing technologies to determine if Borrelia burgdorferi sensu lato is present in ticks and whole blood samples removed from raccoons and opossums trapped in south-central Kentucky. Raccoons and opossums were trapped in Barren and Warren counties of Kentucky between June 2007 and June 2008. Ticks were removed and stored in 70% ethanol. Sterile blood samples were collected into three 10 ml tubes containing the anticoagulant K2EDTA and stored at 4°C. Genomic DNA was extracted from ticks and blood samples using a QIAamp DNA mini kit and a QIAamp DNA blood mini kit (Qiagen) respectively. DNA samples were analyzed by polymerase chain reaction (PCR) for the presence of B. burgdorferi using oligonucleotide primers specific for the OspA gene. A total of 976 ticks were collected. Three different species were obtained from raccoons; Dermacentor variabilis, Amblyomma americanum, and Ixodes sp. Dermacentor variabilis was the only tick species found on opossums. Twenty-five percent (163/642) of the tick DNA samples were positive for Borrelia burgdorferi. Prevalence of B. burgdorferi by tick species was 24.4% (141/577) in D. variabilis, 40.6% (13/32) in A. americanum, and 27.6% (8/29) in I. scapularis. In the present study, 15.7% (8/51) of the total raccoon blood samples examined by PCR were positive for B. burgdorferi, while no opossum blood samples were positive. The high prevalence of B. burgdorferi in ticks common to raccoons and opossums observed in this study, as well as in a tick species that aggressively bites humans in the southeast U. S. (A. americanum), creates concern that there are ample opportunities for people to come in contact with the infected ticks on these animals. Future studies are urgently needed to fully assess the presence and prevalence of B. burgdorferi in Kentucky and other southeastern states in the U. S.

Tick-borne diseases represent major public and animal health issues worldwide. Ixodes ricinus, primarily associated with deciduous and mixed forests, is the principal vector of causative agents of viral, bacterial, and protozoan zoonotic diseases in Europe. Recently, abundant tick populations have been observed in European urban green areas, which are of public health relevance due to the exposure of humans and domesticated animals to potentially infected ticks. In urban habitats, small and medium-sized mammals, birds, companion animals (dogs and cats), and larger mammals (roe deer and wild boar) play a role in maintenance of tick populations and as reservoirs of tick-borne pathogens. Presence of ticks infected with tick-borne encephalitis virus and high prevalence of ticks infected with Borrelia burgdorferi s.l., causing Lyme borreliosis, have been reported from urbanized areas in Europe. Emerging pathogens, including bacteria of the order Rickettsiales (Anaplasma phagocytophilum, “Candidatus Neoehrlichia mikurensis,” Rickettsia helvetica, and R. monacensis), Borrelia miyamotoi, and protozoans (Babesia divergens, B. venatorum, and B. microti) have also been detected in urban tick populations. Understanding the ecology of ticks and their associations with hosts in a European urbanized environment is crucial to quantify parameters necessary for risk pre-assessment and identification of public health strategies for control and prevention of tick-borne diseases.

Les maladies à transmission vectorielle sont dues à des agents pathogènes transmis par des arthropodes hématophages. Ces vecteurs assurent une transmission active (mécanique ou biologique) d’un agent infectieux d’un vertébré vers un autre vertébré. A l’échelle mondiale, les tiques sont responsables de la transmission de la plus grande variété d’agents pathogènes, elles transmettent des microorganismes responsables de maladies bactériennes (borréliose de Lyme, rickettsioses) ou parasitaires (babésioses, theilérioses), ou même virales (encéphalite à tiques).Les Antilles se situent au cœur de la zone Néotropicale des Caraïbes, et constituent une zone à risque pour l’émergence de maladies vectorielles en raison des conditions climatiques favorables aux vecteurs et des échanges intercontinentaux importants (flux illégal d’animaux, oiseaux migrateurs,…). La situation épidémiologique de la zone Caraïbe vis-à-vis des maladies transmises par les tiques est très peu documentée. Les études menées sur le terrain portent essentiellement sur des agents pathogènes affectant les animaux comme Ehrlichia ruminantium, Babesia (bovis et bigemina) et Anaplasma marginale et sont donc loin de pouvoir répondre aux questions concernant le risque d’émergence ou de réémergence de maladies à tique. Ainsi, il est nécessaire et urgent de développer des outils efficaces de surveillance épidémiologique qui permettraient la détection des agents pathogènes, nouveaux, connus ou non suspectés présents dans les tiques. C’est dans ce contexte d’amélioration des performances de veille sanitaire des maladies à tiques dans les Caraïbes que prend place le projet de thèse. La visée de la thèse était de faire un état des lieux des agents pathogènes d’intérêt médical et vétérinaire présents dans les tiques caribéennes à l’aide de techniques de détection à haut débit. Pour cela nous avons d’abord réalisé un séquençage à haut débit d’ARN extraits de tiques collectées en Guadeloupe et en Martinique afin de réaliser un inventaire sans a priori des agents pathogènes (bactéries, parasites, et virus) présents. Cette analyse a permis de mettre en évidence une grande diversité en microorganismes pathogènes au sein de nos échantillons, révélant également la présence de quatre virus appartenant à de nouveaux genres viraux récemment décrits et associés aux arthropodes. Les informations obtenues via le séquençage, additionnées aux données disponibles dans la littérature ont permis de constituer ainsi une liste des agents pathogènes transmis par les tiques nécessitant une surveillance sanitaire dans les caraïbes. A partir de ce répertoire nous avons développé un système de dépistage à haut-débit d’agents infectieux applicable à toute la zone des caraïbes. L’outil de détection est un support microfluidique de type puce à ADN, basé sur la technologie BioMarkTM dynamic arrays (Fluidigm Corporation) qui permet de réaliser de la PCR en temps réel à haut débit afin de détecter simultanément 48 à 96 cibles au sein de 48 à 96 échantillons. Deux puces ont été développées, une première pour le suivi des bactéries et parasites, et une deuxième pour le suivi des virus. Leur performance a été testée sur des échantillons de tiques collectées en Guadeloupe et en Martinique. Ce dépistage à grande échelle a donné un aperçu complet de la situation épidémiologique de 45 bactéries, 17 parasites and 31 virus potentiellement transmis par les tiques dans les Antilles Françaises. La méthode de surveillance développée durant cette thèse représente une amélioration majeure des techniques de veille épidémiologique, permettant la détection rapide et concomitante d’un large panel d’agent pathogène. Elle sera prochainement appliquée au criblage à haut débit des agents infectieux présent dans des tiques collectées à travers la Caraïbe, provenant notamment de Trinité-et-Tobago, Saint-Kitts, la Barbade, et Sainte-Lucie, grâce à la collaboration du réseau CaribVet, et de vétérinaires locaux / Vector-borne diseases are illnesses caused by pathogens transmitted by haematophagous arthropods which provide active transmission (mechanical or biological) of infectious agents from one vertebrate to another. Among these vectors, ticks are known to carry and transmit the greatest variety of pathogens of public health and veterinary importance. They transmit microorganisms responsible for bacterial (Lyme borreliosis, rickettsioses), parasitic (babesiosis, theileriosis), or viral diseases (tick-borne encephalitis).The Antilles are located in the heart of the Caribbean Neotropical Zone. This area can be considered at risk for the emergence of vector-borne diseases mainly due to favorable environmental conditions and intercontinental exchanges (e.g. legal and illegal animal trade, migratory birds). However, the epidemiological situation of the Caribbean area, with regard to tick-borne diseases, is still poorly documented. Indeed, most of field studies only focused on animal pathogens such as Ehrlichia ruminantium, Babesia (bovis and bigemina) and Anaplasma marginale and questions about the risk of emergence or re-emergence of tick-borne diseases remain unanswered. Thus, it is crucial to develop efficient epidemiological surveillance tools that would enable the detection of new, known or unexpected pathogens present in ticks. In this context, the main objective of my thesis was to obtain an overview of pathogens of medical and veterinary interest present in Caribbean ticks using new high-throughput technologies. We first used a high-throughput sequencing approach to determine pathogens present in ticks (bacteria, parasites, and viruses) collected in Guadeloupe and Martinique. This analysis revealed a great diversity of pathogenic agents in our samples and highlighted the presence of four viruses belonging to new viral families recently described and associated with arthropods. Results of sequencing combined with data available in the literature allowed us to make the most exhaustive list of pathogens potentially transmitted by ticks and requiring health surveillance in the Caribbean area. From this pathogen inventory, we developed a system of high-throughput screening of infectious agents applicable to the whole Caribbean area. This molecular tool is a microfluidic system based on the BiomarkTM dynamic arrays technology (Fluidigm Corporation), which enables high-throughput real-time PCR to simultaneously detect 48-96 targets within 48 to 96 samples. Two different chips have been developed, one for bacteria and parasites monitoring, and one for viruses. Their efficiency was tested on tick samples collected in both Guadeloupe and Martinique. This large-scale screening provided a comprehensive overview of the epidemiological situation of 45 bacteria, 17 parasites and 31 viruses potentially transmitted by ticks in the French West Indies. The high-throughput detection tool developed during my thesis represents a major improvement in epidemiological surveillance technology, enabling the rapid and concomitant monitoring of a wide range of pathogens. It will soon be applied to high-throughput screening of infectious agents found in ticks collected throughout the Caribbean, including Trinidad and Tobago, St. Kitts, Barbados, and St. Lucia, thanks to the collaboration with the CaribVet network, and local veterinarians

Cette thèse est une investigation de l'écologie et de l'épidémiologie spatiale fine de la tique à pattes noires (Ixodes scapularis) et du risque associé aux pathogènes transmis par cette tique dans une forêt du sud du Québec, au Canada. Sous l’influence de changements climatiques et environnementaux, la répartition spatiale de cette tique s’étend actuellement à travers la province, et son abondance augmente dans la plupart des régions. Il en résulte une préoccupation croissante en matière de santé publique liée à l'émergence de maladies transmises par les tiques (MTT) dans la province. Ainsi, plusieurs besoins de recherche ont vu le jour, y compris l’amélioration de la compréhension des facteurs dictant où et quand le risque émergera, et l’identification des composantes sur lesquelles les autorités régionales, locales et les individus pourraient agir afin réduire ce risque. Le site d’étude pour ce projet était le parc National du Mont-Saint-Bruno. De 2016 à 2018, à 32 sites répartis dans les habitats forestiers du parc, les tiques ont été collectées par la technique de la flanelle, la communauté d’hôtes de la tique a été inventoriée (grands et petits mammifères, et oiseaux nicheurs) et les micro-habitats ont été caractérisés (en termes de structure, de composition et de conditions microclimatiques). La densité des tiques dans la zone d’étude était particulièrement élevée par rapport aux autres régions environnantes. Les analyses spatiales ont montré des patrons d’abondance des tiques, persistants sur trois ans, avec un effet significatif des conditions locales de températures et d’humidité relatives. Un effet significatif de la densité du stade de développement précédent lors de l’année précédente a également été noté, suggérant une dispersion lente des tiques par les hôtes et une dynamique d’invasion caractérisée par plusieurs évènements d’introduction et d’expansion opérant à une échelle locale. En 2017 et 2018, un dispositif d’échantillonnage plus extensif a visé la communauté d’hôtes des stades immatures d’I. scapularis, afin de documenter le rôle de ces espèces dans la circulation des agents pathogènes. 849 oiseaux nicheurs appartenant à 50 espèces et 694 petits mammifères appartenant à 4 espèces ont été examinés pour la présence des tiques et des pathogènes. Les souris à pattes blanches (Peromyscus leucopus) contribuaient à la majorité (80%) des cas d’infection des nymphes par Borrelia burgdorferi dans l’échantillon, alors que les 20% restants étaient attribuables à des oiseaux. Ce résultat confirme la proposition de certains auteurs à savoir que l’importance des hôtes alternatifs (autres que P. leucopus) aient été sous-estimée dans les études précédentes, et souligne l’importance de poursuivre les travaux pour documenter l’impact de ces hôtes dans l’épidémiologie des MTT. Finalement, la caractérisation des patrons de risque associés aux MTT dans la zone d’étude a été complétée par un échantillonnage de l’intensité d’utilisation des sentiers par les visiteurs du parc en 2017 et 2018. D’abord, les prélèvements de tiques dans l’environnement et sur les hôtes ont permis de détecter la présence de trois pathogènes représentant un risque de santé publique pour la population locale, soit en ordre du plus au moins prévalent : B. burgdorferi, Anaplasma phagocytophyllum et Borrelia miyamotoi. Ensuite, l’intégration des patrons spatiaux de l’utilisation du parc par les visiteurs a mis en évidence le rôle des facteurs comportementaux des utilisateurs et des attributs du paysage local dans la détermination des patrons de risque de contacts entre les visiteurs et les nymphes infectées par B. burgdorferi. Le risque de contact était associé à certaines périodes et zones de forte fréquentation du parc, mais pas aux indicateurs locaux de fragmentation de l'habitat, comme le suggèrent d'autres études. En outre, les zones où la densité de la couverture forestière et des points d'intérêt (infrastructures) étaient les plus élevés présentaient les niveaux de risque les plus élevés. Cette thèse contribue à l’avancement des connaissances fondamentales reliées à la biologie des tiques, aux relations écologiques entre ces ectoparasites et leurs hôtes et à l’écologie des pathogènes qu’elles transmettent. Ces connaissances trouveront de multiples applications concrètes, principalement dans le domaine de la santé publique où elles permettront notamment de parfaire les outils existants d’évaluation du risque et de prévention des MTT. En reliant l’écologie à la santé publique, cette thèse répond à un besoin de recherche et de développement d’expertise qui devient de plus en plus important dans le contexte actuel des changements climatiques et de l’émergence des maladies zoonotiques qui s’en trouvent facilitées au Canada. / This thesis is an investigation of the ecology and fine-scale spatial epidemiology of the blacklegged tick (Ixodes scapularis) and the risk associated with pathogens transmitted by this tick in a forest of southern Quebec, Canada. Under the influence of climate and environmental changes, the spatial distribution of this tick is currently expanding across the province, and its abundance is increasing in most regions. This has resulted in a growing public health threat associated with the emergence of tick-borne diseases (TBDs) in the province. Thus, several research needs have emerged, including improving understanding of the factors determining where and when risk will arise, and identifying what regional and local health authorities and individuals can do to reduce that risk. The study site for this project was Mont-Saint-Bruno National Park. From 2016 to 2018, at 32 sites distributed in the park's forest habitats, ticks were collected by drag sampling, the host community was inventoried (large and small mammals, and nesting birds), and microhabitats were characterized (in terms of structure, composition, and microclimatic conditions). The density of ticks in the study area was particularly high compared to other surrounding areas. Spatial analyses showed patterns of tick abundance, persistent over three years, with a significant effect of local microclimatic conditions (relative temperature and humidity). A significant effect of the density of the previous developmental stage in the previous year was also noted, suggesting slow host dispersal of ticks and invasion dynamics characterized by several introduction and expansion events operating at a local scale. In 2017 and 2018, a more extensive sampling scheme targeted the host community of immature stages of I. scapularis, to document the role of these species in pathogen circulation. 849 breeding birds belonging to 50 species and 694 small mammals belonging to 4 species were examined for the presence of ticks and pathogens. White-footed mice (Peromyscus leucopus) contributed to the majority (80%) of Borrelia burgdorferi nymph infections in the sample, while the remaining 20% were attributable to birds. This result confirms the suggestion by some authors that the importance of alternative hosts (other than P. leucopus) may have been underestimated in previous studies and underscores the importance of further work to document the impact of these hosts in the epidemiology of TBDs. Finally, characterization of risk patterns associated with TBDs in the study area was completed by sampling trail use by park visitors in 2017 and 2018. First, the analysis of ticks collected from the environment and on hosts demonstrated the presence of three pathogens representing a public health risk to the local population, in order from the most to the least prevalent: B. burgdorferi, Anaplasma phagocytophyllum and Borrelia miyamotoi. Second, integration of spatial patterns of visitor use of the park highlighted the role of visitor behaviour and local landscape attributes in determining patterns of risk of contact between visitors and B. burgdorferi-infected nymphs. Risk was associated with certain times and areas of increased park use, but not with local indicators of habitat fragmentation, in contrast with the results of previous studies. In addition, areas with the highest densities of forest cover and points of interest (infrastructure) had the highest levels of risk. This thesis contributes to the advancement of fundamental knowledge related to the biology of ticks, the ecological relationships between these ectoparasites and their hosts, and the ecology of the pathogens they transmit. This knowledge will have multiple applications, mainly in the field of public health, where it will allow the refinement of existing tools for risk assessment and prevention of TBDs. By linking ecology to public health, this thesis responds to a need for research and development of expertise that is becoming increasingly important in the current context of climate change and the emergence of zoonotic diseases that it facilitates in Canada.